Changes in synthesis of DNA-binding proteins during the onset of transformation in NRK cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

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CopyrightX)1977 AmericanSociety for Microbiology Printed inU.S.A.

Changes

in

Synthesis

of DNA-Binding

Proteins

During

the

Onset

of

Transformation in NRK Cells Transformed by a

Temperature-Sensitive

Mutant of Rous Sarcoma Virus

BRUCE E. MAGUNI* AND PRESTON H. DORSETT

Department ofAnatomy, University of Tennessee Center for the Health Sciences, Memphis, Tennessee38163,

and Department ofMicrobiology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163

Received for publication 23 November 1976

Synthesis

of cytoplasmic DNA-binding proteins was investigated after a shift

from the

nonpermissive to the permissive temperature in NRK cells

trans-formed

by a

temperature-sensitive mutant of Rous sarcoma virus [ts339(RSV)].

Cells were labeled for several generations in [3H]leucine and were

pulse-labeled

with

['5S]methionine

for 1 h at the

nonpermissive temperature

(390C)

and at the

permissive

temperature

(330C,

5 h after

shift from

390C).

Proteins binding to

sequential columns of

double-stranded and single-stranded DNA-cellulose were

examined by polyacrylamide gel electrophoresis in the presence of sodium

dodecyl sulfate, and the 5S/3H ratios were obtained for

each

column fraction

and

for individual

polypeptides. The protein fractions binding to single-stranded, but

not

double-stranded, DNA and eluting at high salt concentrations (greater than

0.60

M

NaCl) showed elevated 35S/3H ratios. This

indicated increased synthesis

of these

proteins

within 5 h after the onset

of

transformation. The

majority of the

polypeptides in these fractions showed

increased synthesis as a consequence of

transformation. One prominent polypeptide among them

constituted

0.1%

of

the

cytosol

protein

and had a

molecular

weight

of

93,000. We conclude that the

synthesis of

proteins

binding

tightly

to

single-stranded

DNA

is

increased

early

after

the onset of transformation.

Cells

transformed

by

temperature-sensitive

(ts)

transformation

mutants

of Rous sarcoma

virus

(RSV) permit the study of sequential

events

that

occur

during

the course of

transfor-mation

(3, 11, 13, 14, 20, 27). Most

of these

studies have

focused

on

early

membrane-re-lated changes

that occur during the onset of

transformation in such systems.

Recent

experimental data

have

raised

the

possibility

that

viral-coded DNA-binding

pro-teins may

be

involved

in

the process

of

transfor-mation.

Cells

infected or

transformed

by

hu-man

adenovirus

types 2

and 5

produced

two

new

DNA-binding polypeptides (25, 21); Tenen

et

al.

(29)

demonstrated

that the

thermosensi-tive

defect

in

a ts

A-type mutant

of simian virus

40 was in

the T

antigen. This

DNA-binding

protein (6, 12, 22, 26) attaches at or

near

the

replication point of simian virus

40

DNA

(22).

Although

a

functional

relationship between

transforming

ability

and

DNA-binding

capac-ity of

the

T

antigen has

not

yet been

demon-strated,

the

possibility exists that the initial

'Present address: DepartmentofAnatomy, University

ofArizona,CollegeofMedicine,Tucson,AZ 85724.

action

of

the

transforming

gene

product

occurs on

the

cellular DNA.

DNA

affinity

chromatography has been

used

in

the

purification of procaryotic and eucaryotic

proteins whose

DNA-related

roles have been

elucidated

(1, 4, 10, 16, 23, 25, 30, 31).

It also has

been

useful

in

selecting proteins that may

func-tion in DNA

replication, repair, or

recombina-tion (8).

The

genomes

of T-even

phage

and

herpes simplex

virus

contain a

high proportion

of genes involved with DNA

replication

(19, 23,

28)

and apparently code for a large number of

DNA-binding proteins (5, 10, 21). Hence, DNA

affinity chromatography

has

permitted

the

se-lection, without prior knowledge

of

their

func-tion,

of a group of proteins

that may function

in

aspects

of

DNA

replication.

The aim of the

present investigation

was to

examine

alterations

in

the

synthesis

of

DNA-binding proteins after

a

shift from the

nonper-missive to

the

permissive temperature,

ina rat

cell

line

(NRK)

transformed by

a ts mutant

of

the

B77 strain

of

RSV,

ts339(RSV)

(9). These

cell cultures were incubated with radioactive

amino

acids from

h 5

to

6

after

temperature

469

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470 MAGUN AND DORSETT

shift. The

incorporation

of radioactivity

into

DNA-binding proteins of shifted cultures was

compared

with that obtained from cultures

which had been

treated similarly,

but which

had

been

maintained

at

the

nonpermissive

temperature.

The results of this

investigation

indicate

that the onset of transformation is

ac-companied

by an increased incorporation of

la-bel

into a

small

portion

of the total

DNA-bind-ing

protein complement and that these proteins

bind

to

single-stranded DNA,

but not to

double-stranded DNA.

MATERIALS AND METHODS

Cells and culture. NRK cells and NRK cells transformed by ts339(RSV) wereobtained from W. K. Joklik, Duke University. The thermosensitive transformed cell line [ts339(RSV)NRK] had been established from a clone of transformed cells in soft agar (9).The cell lines werepropagatedinDulbecco minimal essential medium containing 10% heat-in-activated(560C, 30min)fetalcalf serum. The cells wereroutinelypropagatedasmonolayer cultures in 75-cm2 flasks.

To obtain sufficient cells, roller bottle cultures were utilized. Roller bottles (685 cm2) were seeded

with 1 x 107to 2 x 107ts339(RSV)NRKcells. NRK

roller cultures were prepared byseeding plastic bot-tles (490cm2)with5x 107to 10 x 107cells. Since the NRK cells did not attach well at390C, allcultures wereincubated for the initial 18 to 24 h at360C and then incubated at 390C. The culture fluid was

changedat 24- to 48-h intervals; thestatus ofthe

monolayer was monitored microscopically each day. Afteraconfluent monolayerhad beenobtained, the cultures were incubatedanadditional 48 h without

feeding to establish contact inhibition. The

ts339(RSV)NRK cells routinely established a

con-fluent monolayer within 3 to 4 days, whereas the

NRKcells routinelyrequired 6 to8days.

Cell culture reagents were purchased from Micro-biological Associates, Rockville,Md.

Preparation of cytosol. Cells were harvested by scraping, washed twice in cold phosphate-buffered

saline at low speed, and allowed to swell for30min in 2volumes of hypotonic buffer (1 mM MgCl2, 0.4

mM CaCl2, 0.5 mM dithiothreitol, 1 mM

phenyl-methylsulfonyl fluoride, and 25mM Tris-hydrochlo-ride, pH 7.9) at

00C.

The cells weredisrupted in a Douncehomogenizer with a "B" pestle, after which 2 volumesof0.05 MNaCl,0.1%Brij 58,1mM phenyl-methylsulfonyl fluoride, 1 mM EDTA, 10 mM /3-mercaptoethanol, 10% glycerol, and 10 mM Tris-hydrochloride, pH 8.1 (buffer A) was added. The homogenatewascentrifuged at 25,000xg for 15min and again at 100,000 x g for 1 h, after which the supernatantwaswithdrawn and made to contain 1.7 MNaCl. Polyethylene glycol-6000 was added to a final concentration of 10% for precipitation of DNA. Afterincubationat0°Cfor30min, the samples were clearedby centrifugation at 7,400 x g for 15 min. The resulting supernatant was dialyzed against threechanges of50volumesof buffer Aat4°C. The

supernatant that formed after centrifugation at 20,000 xgfor 20 min wasdesignated as the cytosol fraction.

Preparation of DNA-cellulose columns. Native and denatured DNA-cellulose preparations were made bythe combined methods of Litman (16) and Alberts and Herrick (2). To prepare native DNA-cellulose, 50 mg of calfthymusDNA (Worthington

Biochemicals,Freehold, N.J.)wasdissolvedin 18ml of buffer B (0.01 MTris-hydrochloride, 1mMEDTA, pH 7.4).Six gramsof Cellex410(Bio-Rad Laborato-ries, Richmond, Calif.) was added, and the slurry

wasdriedat45°Covernight. The dried DNA-cellu-lose was pulverized and resuspended in ethanol,

followed byUV irradiation, as described (10). The dried native DNA-cellulose wasswollen for 2 h in buffer Bat4°Cand then washedbycentrifugation several timesinbuffer A before being packed into columns.Denatured DNA-cellulosewasprepared by heating 1.3 mg of calf thymus DNA per mlin0.05M NaOHat100°C for 10min,followed by rapid chilling

in ice water.After the pH had beenadjustedto7.2,1 gof Cellex 410wasaddedtoeach3mlof denatured DNA, andthe slurry was dried overnight at room temperature. The DNA-cellulosewas mixed again with denatured DNA and driedasbefore. The dena-tured DNA-cellulose was resuspended in buffer A and washed several timesbycentrifugationand

re-suspension. Native and denatured DNA-cellulose contained atleast 2 mg of DNA perpackedml of columnvolume. Swollen DNA-cellulose (denatured ornative)waspackedinto glasscolumns (0.7by 8 cm)containingavolume of3ml.Native DNA-cellu-lose was overlaid with 1 ml of plain Cellex 410, whichwasremoved before elution of bound protein toremoveproteinthat hadboundnonspecificallyto cellulose before elution of thespecifically bound pro-tein.

DNA-cellulose chromatography. Thirty to forty

milliliters of cytosol in buffer A was pumped

through the column ofnative DNA-cellulose at a

flowrateof12 ml/h.After it had beenloaded, the

column was flushed with 17 ml of buffer A. The upper plain cellulose pad was removed, suspended

in 5mlof water,andassayed for radioactivityinan aqueouscocktail. Less than1%of the total column-bound protein was adsorbed to the plain cellulose

pad. The native DNA-cellulose column was then eluted with successive 10-ml volumes of buffer A containing 0.1, 0.15, 0.25, 0.60, and 2.0 M NaCl. Eluateswereconcentratedto 1 ml in an ultrafiltra-tiondevice (Amicon MMC) containingaPM10 filter. Five percentsodium dodecyl sulfate (SDS), contain-ing5%

/3-mercaptoethanol,

wasaddedtothe concen-trated sample, which was then heated in boiling waterfor1minanddialyzed against electrophoresis sample buffer (0.01Mphosphate, pH 7.2,0.1%SDS,

1% mercaptoethanol). Theradioactivity remaining inthe column afterthe 2.0 Msalt elutionwas mea-suredbysuspendingthecolumncontents indistilled waterand assayingaportion forradioactivity. All unretained cytosol from the native DNA-cellulose column was then pumped through a similar-sized column packed with denatured DNA-cellulose,

washed, and elutedasdescribedfor the native

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DNA-BINDING PROTEINS AND

471

cellulose columns. Eluted fractions were concen-trated and dialyzed against electrophoresis sample buffer.

Scintillation spectrometry. Aqueous samples

wereadded to 10 ml of scintillation mixture, 1 part Triton X-100, and 2 parts toluene containing 2,5-diphenyloxazole (4.9 g/liter) and bis-MSB (0.1 g/ liter). Gels to be assayed for radioactivity were fro-zen ondryiceand stored at -20'C. The gels were sliced transversely into1-mmwafers with a Hoefer gel slicer (15 cm long). Each slice was incubated overnight with0.5 mlof hydrogen peroxide at450C

in atightly capped polyethylene vial. After 24 h, each vial received the scintillation cocktail and was counted in a scintillation spectrometer (Beckman LS230). Under theseconditions,3Hefficiencywas at least 22%. 35Sand 3H activity were determined

si-multaneously by using the channels ratio method with an externalstandard.

Protein determination. Protein was determined according to Lowry et al. (17) with bovine serum albumin asstandard. Because of the small amounts of proteinin the DNA-binding protein fractions, it wasnecessary to measure protein content bythe use

ofradioactivity. Allcultureswere incubated for at

least six generations in[3H]leucine (1.0

,.tCi/ml).

A small amount of cytosol protein was dialyzed against 0.1 N NaOH and assayed both for protein and tritium. Withthese conditions, 800 dpm of 3H per

jig

of protein wasobtained, and this valuewas foundtobereproducible within 5% amongseveral experiments. Because of the small amounts of pro-tein used for electrophoresis, radioactivity assay proved to be the most accurate means of measuring protein concentration and was applied throughout this investigation.

Polyacrylamide gel electrophoresis. Protein frac-tionsinsample bufferwereexaminedby polyacryl-amide gelelectrophoresis (PAGE)inthe presence of SDS accordingtoLaemmli(15). The separationgel was 12.5 cmlong and contained 8.75% acrylamide and 0.23% methylenebisacrylamide. Molecular-weight marker proteins were as previously de-scribed (18).

RESULTS

Phenotypic

changes after

temperature

shift. The experiments described

in

the

present

investigation are

based

on

the induction of the

transformed

state

by alteration of

incubating

temperatures

from the nonpermissive

(390C)

to

the

permissive

(33°C)

temperature. It was

nec-essary

first

toexamine

cellular

properties

phe-notypic

of transformation

as a

temporal

conse-quence oftemperature change.

When grown at

39°C,

the

ts339(RSV)NRK

cells formed

a confluent

monolayer

similar to

that of nontransformed cells

(Fig.

1). After the

temperature was

shifted

to

33°C,

the cells

as-sumed a

transformed

phenotype,

which was

evident as

early

as 6 h after the temperature

shift

(Fig.

ic). Cells migrated

intoclusters

and

acquired

arounded morphology instead of the

flat, stellate shape that

was

characteristic

of

their

growth

at 39°C.

Twenty-four

hours after

shift to

the

permissive

temperature,

the

trans-formed appearance was seen in

its

extreme

form. Cellsthat had

been

maintained

at

39°C

throughout the

course

of the experiment

main-tained

their untransformed

appearance

(Fig.

lh).

As

ts339(RSV)NRK

cells

approached

con-fluency

at

39°C, their

rate

of

DNA

synthesis

apparently

decreased,

as

demonstrated by

the

reduction in

incorporation

of

[3H]thymidine

into acid-precipitable

material

(Fig.

2).

How-ever, if

parallel cultures

were shifted to

33°C,

the

incorporation

of

[3H]thymidine began

to

in-creasewithin 6 h and continued to rise over a

24-h

time period.

It

thus

appeared that a shiftto

thepermissive temperature was able to

stimu-late DNA replication of

ts339(RSV)NRK

cells

within a few hours after the temperature

change.

Fractionation

of

DNA-binding

proteins.

DNA-binding

proteins

wereobtained by

perco-lation of cytosolthrough sequential columns

of

native

(double-stranded)

and denatured

(sin-gle-stranded) calf thymus

DNA-cellulose,

after

which the bound proteins wereeluted

stepwise

with

increasing

salt

concentrations. Elution

profiles of the

DNA-binding

proteins from

na-tive DNA showed

discrete

peaks of protein,

which were removed by each successive salt

wash(Fig. 3A). The 2.0 M NaCl wash contained

little

detectable

protein.

The

native

DNA-cellu-lose column bound approximately 4% of the

total

cytosol protein,

96% ofwhich was

subse-quently recovered

in

the NaCl

eluates

(Table 1). An additional 4% of the cytosol protein

ad-sorbed

to

denatured DNA,

and 90%

of

these

proteins were

recovered

in

the NaCl

eluates

(Fig.

3B, Table 1). In contrast to the elution

profile from native

DNA-cellulose, the

2.0 M

NaCl eluate from denatured DNA

comprised

a

sizable peak.

Approximately

half of the

pro-teinsbinding to denatured DNA were removed

in

the

0.10 M

NaCl

wash.

The sizes of the

DNA-binding protein fractions

were

similar,

whether

cells had been grown at

39°C

or had been

shifted to

33°C

for 6 hbefore

harvesting.

PAGE

of

DNA-binding proteins. The

DNA-binding proteins

in

each

NaClfraction

(except

the 2.0 M NaCl wash from native

DNA,

which

contained insufficient

protein

for

characteriza-tion)

were examined

by

PAGE. Each fraction

wasfound to contain a

unique

polypeptide

frac-tion

when

compared

with

either

the other

DNA-binding

protein

fractions

or the

unfrac-tionated

cytosol

proteins (Fig. 4).

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472

MAGUN AND DORSETT

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FIG. 1. Morphological changes ints339(RSV)NRK cells afterashift from 39to33°I. Slide culturesof

ts339(RSV)NRK cells were incubated at 39°C until a confluent monolayerwasobtained,after whichsomeof

the cultures were shifted to

33XC

and some remained at390C. Slide cultureswerefixed and stained with hematoxylin and eosin at the time ofshift to33XC(a), and at 3 h (b), 6 h (c), 9 h (d),12h (e),24 h(I), and 48h (g)after the shiftto33C.Similar culturesmaintainedat39°Cwerealsofixedandstained at thesametimes. These cultures all appeared similar to those in(h), which showsacellculture maintainedat39°Cthroughout

the 48-h experiment.

pared

with the unfractionated

cytosol proteins,

most

of the DNA-binding proteins

were

rich

in

high-molecular-weight

polypeptides

(larger

than 80,000). Comparison of cytosol proteins

before

and

after

DNA-cellulose

chromatogra-phy

(Fig. 4)

showed

few changes, indicating

that the

DNA-binding

polypeptide

profiles

rep-resented

an

amplified

view

of minor

compo-nents present in

the

whole cytosol. Although

polypeptide profiles obtained

from cells

shifted

from 39 to

330C

for 6 h

showed

some notable

differences when

compared with

profiles of

pro-teins

obtained from cultures

maintained

at

3900,

the

profiles obtained

at

both

tempera-tureswere

remarkably similar.

Incorporation

of

labeled

amino acids into

DNA-binding

proteins.

If

changes

in the

syn-thesis of

DNA-binding proteins

occur

after

a

shift

to

the

permissive temperature,

it

might be

expected

that incorporation

of radioactive

amino

acids

into

those DNA-binding proteins

also

would

reflect changes. To

test

this

hypoth-esis,

twosets

of

ts339(RSV)NRK cells

were

in-cubated with

[3H]leucine

at

390C

for six

genera-tions

to

permit uniform incorporation

of

[3H]leucine

into

cellular protein. One

set

of

these

cultures

was

pulse-labeled

with

[35S]methionine

from h

5 to 6

after

a

shift

to

330C.

The

35S/3H

ratios

of

the

DNA-binding

pro-teins

synthesized

after the shift

to

33WC

were

compared

with

the

35S/3H ratios

of

DNA-bind-ing proteins

from the other

set

of cultures

that

had been

maintained

at

390C

and

incubated

with

[35S]methionine

for 1 h.

Comparison

ofthe

I5S/3H

ratios

from

the two

experimental

groups,

(35S/3H

at

330C)/(35S/3H

at

3900),

referred

to

henceforth

asR,

should be

an

indicator of

spe-cific

protein fractions

whose

rate

of

synthesis

becomes altered after

a

temperature shift.

In the

ts339(RSV)NRK

cells, a shift from 39 to

330C

resulted

in

decreased

R

values,

ranging

from 0.90 to 0.98for the

cytosol

and

DNA-bind-ingprotein fractions, except for the 0.60

and

2.0

MNaCl

eluates

from

denatured

DNA

(Table

2).

Thelatter fractions had

elevated

Rvalues(1.36

and 1.23,

respectively),

indicating

that,

inthese

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5 10 15

T I M E (hrs)

FIG. 2. Synthesis of DNA in ts339 cellsafterashift fromthe nonpermissil

missive temperature. ts339(RSV)NRK seeded in 60-mm culture plates and i 360C for12to18h.The culturesthenwe at39'C until themonolayers had become Fourteen hours afterthe culture medium replacedwithfreshmedium, one-halfof wereshiftedto33'C.Atthetimeofthe shift(zero time)andat1,2,3, 4, 5,6,E and 24 hafter thetemperatureshift,fou 33and390Cwereincubatedwith

[3H]thy

h. [3H]thymidine (5 uCi) in 0.1 ml o) buffered saline was addeddirectlyto e After the 1-h pulse-labeling period, th scraped from the dishes andpipetted i chloroacetic acid. Theacid-precipitabler collectedonglass-fiber filters, andthe; incorporatedperculturewasdetermined represents theaverage ofthefourculture turemediumwasnotchangedthroughout

fractions,

therewas anetincreased

tion of35S into protein after the to

shift.

In an attempt to determine whet

creased R values of the 0.60 and 2

fractions from denatured DNA occ

consequence ofthe onset oftransfc

thets339(RSV)NRK cells,asimilar

was performed with NRK cells th

been transformed by RSV. All DI

protein fractions showed decreased

ranging from 0.46 to0.65 (Table 2).

tothe increased R values of the0.6(

NaCl fractions from denatured DN

from the ts339(RSV)NRK cells, t

fractions inthe NRKcells exhibited

R values (0.46 and0.48). Since the

R valueisprobablyaconsequencea

enzymaticactivitydue to the lowerE

ture, the increaseinR value in the

ing protein fractions from thets339

cells after temperature shift should

increased synthetic

rates

of

someor all ofthe

439 proteins inthese fractions.

433 Isotopic labeling of individual DNA-binding

polypeptides. The DNA-binding protein

frac-tionsfrom the ts339(RSV)NRK cellsinthe

tem-perature shift experiment shown in Table 2

were analyzed by PAGE. Each gel was sliced

transversely into 1-mm slices, and each slice

wasanalyzed for 35S and 3H activity. The

35S/3H

ratioswereobtained for corresponding

polypep-tide bands from shifted and nonshifted

cul-tures, and anR value wascalculated for each

pair of correspondingbands.

20 2 5 The R values ofcorresponding bands ofthe

electropherograms

are shown in Fig. 5for the

?(RSV)NRK 0.60 and 2.0 M eluates from denatured DNA.

'eto theper- The R values formostbands in these

fractions

cells were were greater than 1, indicating that an

in-tncubated

at creased incorporationof 355 intothese

polypep-!reincubated tides occurredafter the shiftto330C.Two poly-se confluent. peptides, which exhibited elevated R values in

thecultureens

Fig.

5,

were

present

in

relatively large

temperature

amounts.

One,

which hadan

apparent

molecu-,p10,

12,18 lar

weight

of

35,000,

was

present

in the

0.25,

orculturesat 0.60, and 2.0 M eluates from denatured DNA

vmidinefor1 (Fig. 4)andcomprisedabout 0.75% of thetotal

f

phosphate- cytosol protein. The other major band, which

'ach culture. hada molecular weight of 93,000, was eluted Le cells were from denatured DNAby2.0 MNaCland

com-nto cold tri-

prised

about 0.1% of the total cytosol protein.

materialwas ItsR valuewas1.38.

radioactivity

Corresponding

bands of

other

DNA-binding

'es. The

cupl

polypeptides

were

similarly

analyzed

for the

ttthe36h. native and denatured

DNA-binding

protein

fractions. Some DNA-binding polypeptides

ap-parently increased in quantity after a shift to

lincorpora-

thepermissive temperature (Fig. 4); whenthe

temperature R values of these polypeptides were

deter-mined, allwerefoundtobeless than 0.90 (data

other the in- not shown),

indicating

that the appearance of

.0 M NaCl these bands was probably not due to an

in-urred as a crease in their synthesis. The results for all

rmation in

DNA-binding

protein fractionsaresummarized

experiment in Fig. 6. The

histogram

of R values for 159

Lat had not

DNA-binding

polypeptides (as many as could

NA-binding

be measured accurately), demonstrates that

I R values, most

DNA-binding

polypeptide bandR values

Incontrast were less than 1, with a mean of 0.90. The

0 and 2.0 M majority ofpolypeptides in Fig. 6 with R values

[A obtained greaterthan1.2werecontained in the 0.60 and

these same 2.0 M NaCl elutions from denatured DNA

d decreased (shown inFig. 5).

decrease in Uptakeof

[35S]methionine

atnonpermissive

fdecreased and permissive temperatures. Alterations in

adtempera- the

35S/3H

ratios ofproteins could resultfrom

DNA-bind- temperature-related changes in the uptake of

l(RSV)NRK 35Sintothecells. If this didoccur,the R values

d be due to might indicate an altered availability of

o-- 39-INCORPORATION

OF 3H-THYMIDINE

, 0 0

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I-J It a.

FRACTION NUMBER

FIG. 3. DNA-cellulose chromatography of cytosol proteins.Chromatography of50mgofcytosolprotein wasperformed sequentiallyonnative(a) and dena-tured(b) DNA-cellulose columnsasdescribedin Ma-terials andMethods. ts339(RSV)NRK cells had been incubated in 1.0 ,Ci of[3H]leucineperml foratleast six generations to uniformly label protein. The

amount of protein in each 1-ml fraction was mea-suredby scintillation counting using 800 dpm of 3H per pg of protein as a correction factor. At each

arrow,the molarity of the elution buffer (buffer A)

TABLE 1. Fractionation of DNA-binding proteins from ts339(RSV)NRK cellsgrownat39 and330Ca

NaCl Total

pro-Temp concnof Cytosol tein

Temp elution protein boundto

(OC) buffer (%)b column

(M) (%)

Native DNA-cel-lulose

39 0.10 1.19 27.8

33 0.10 1.22 26.7

39 0.15 1.19 27.8

33 0.15 1.32 28.6

39 0.25 1.07 25.1

33 0.25 1.18 25.8

39 0.60 0.58 13.5

33 0.60 0.59 13.0

39 2.0 0.09 2.1

33 2.0 0.10 2.2

Denatured DNA-cellulose

39 0.10 2.17 47.8

33 0.10 2.23 46.0

39 0.15 0.46 10.0

33 0.15 0.54 11.9

39 0.25 0.62 13.7

33 0.25 0.66 13.6

39 0.60 0.57 12.5

33 0.60 0.56 11.6

39 2.0 0.33 7.3

33 2.0 0.38 7.8

a DNA-cellulosechromatography wasperformed

asdescribed in Materials and Methods withcytosol protein extracted from ts339(RSV)NRK cells cul-tured eithercontinuouslyat390Corshifted for 6h to 330C beforeharvesting.

IProtein contentwasmeasured by scintillation spectrometry asdescribed in Materials and Meth-ods. Fifty milligrams ofcytosol proteinwasloaded ontocolumns.

cTotal proteinwascalculatedasthe quantity of proteinrecovered in thecombined salt fractionsplus theproteinthat remained onthe column after the last salt wash (see MaterialsandMethods).

wasincreasedasshown. Allfractions between suc-cessive arrows werepooled for analysis by PAGEas

describedin MaterialsandMethods. The fractions elutedafter application ofthe 2.0 MNaCl bufferto

denatured DNA-cellulose were also analyzed by PAGE.

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PROTEINS ELUTED FROM

NATIVE DNA

0.10 0.15

in..

-I

0 m

son

vmwl

-!

m

_wmm_

IS-MA

0.2 5 0.

_

_...a-_._

_~

a

A B A B A B A

.60

A.

PROTEINS ELUTED FROM

DENATURED DNA

0.10 0.15 0.25 0.60

_ -_

mho

50

B A B ARB ARB A

2.0

Ili

22 A B

FIG. 4. Electropherograms of cytosolandDNA-bindingprotein fractions. ThecytosolandDNA-binding protein fractions fromts339(RSV)NRK cellseithermaintainedat390C (A)orshiftedto330C (B) for 6hwere examined byPAGE in thepresenceofSDS. Proteinfractionsfrom the experiment shown in Table1 were

concentratedand analyzed by PAGE. Samples containedfrom20to 70 pgof proteinpergel. The cytosol

fractionwasexaminedbefore (X)andafter (Y)the DNA-binding protein had been removed chromatographi-cally. (A)and(B) ofeachpair of gelscontain identicalamountsof protein.

[35S]methionine

rather than altered

synthesis

of proteins. Since identical concentrations of

[35S]methionine existed in the culture fluid

afterthe1-hlabeling periodatboth 39and 330C

(6 h after temperature shift), it appears that

entrance of35S into ts339(RSV)NRK cells was

unaffectedby the temperature shift (Table 3).

DISCUSSION

The use of the ts339 mutant of RSV in a

mammalian cell system offers several

advan-tages over the use of the wild-type virus in

avian cells tostudy transformation (see

refer-ence 9). These are: (i) host cells show relative

genetic homogeneity, (ii)in most cases,

trans-formedcells donotproduce viral particles, and

(iii) normal(untransformed) cells donotappear

tocontainendogenous avian-sarcoma leukosis

viralgenes. Anadditionalvalue of thissystem

is the opportunity to study early molecular

changes during the onset of transformation

afteratemperature shift.

Among the

cytosol

proteins of

ts339(RSV)-NRKcells, those that haveanaffinity for DNA

were found to comprise a small subset, and

mostcouldnotbe detectedamongthe profile of

total cytosol

polypeptides

on SDS gels.

Chro-matographyonnative and denatured calf

thy-mus DNA-cellulose separated these proteins

intoseveral hundred polypeptides. An

accumu-lation ofbiochemical and genetic evidence has

indicated thatmanyDNA-bindingproteinscan

be associated with DNA-related functions in vivo, and that nonspecific (ionic) interaction

ofprotein withDNAis minimizedatthe NaCl

concentrations used for protein adsorption (2).

Although it remains unknown which

DNA-binding polypeptides separated in the current

investigation do in fact exhibit DNA-related

functions within thecell, the method of

analy-sisappearstobeafeasiblemeanstoselect and screen an assortmentofcandidate proteins for

their involvement in early aspects of growth

andtransformation.

CYTOSOL

_ _.

NE!

._

_

X Y

150

K

0

r-80 m 0 c

60x

-4 40

130

0

a~

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TABLE 2. Fractionationof DNA-bindingproteins

from thecytosolofts339(RSV)NRK and NRKcells atthepermissiveand nonpermissive temperature

R Elution

Cell type buffer

Native

Dena-(M)

DNAb

tured

DNAc

ts339(RSV)NRK 0.10 0.93 0.97

0.15 0.94 0.90

0.25 0.93 0.97

0.60 0.98 1.36

2.0 ND 1.22

NRK 0.10 0.53 0.63

0.15 0.49 0.65

0.25 0.56 0.57

0.60 0.55 0.48

2.0 ND 0.46

a Roller bottle cell cultures [either

ts339(RSV)-NRKorNRK]weregrownat390Cfor6daysin 50ml of medium containing50

kuCi

of[3H]leucine per cul-ture, after which one-half of the cultures were shiftedto330C. Five hoursafter temperatureshift,

the medium wasreducedto 5mlperbottle, and150

ACi

of [35S]methionine was added to the medium

containing[3H]leucine. The cultures were pulse-la-beled for1 hbefore harvesting. The other cultures were treated identically, but were maintained at

390C

throughout all labelingprocedures. Acytosol fraction was obtained from each group of cultures and was subjected to DNA-cellulose chromatogra-phy. Analiquot of each column fractionwas ana-lyzed for both3Hand35Sactivity. Each samplewas countedto a minimumof 25,000accumulatedcounts ineach channel.

bR = (35S/3Hat

3300)/(35S/3H

at39CC)forcolumn fractions eluted from native DNA-cellulose. For

ts339(RSV)NRK cytosol,R =0.95; for NRKcytosol,

R =0.55.

cR was determined for column fraction eluted fromdenatured DNA-cellulose.

dND, Notdone.

The results presented in this paper

demon-strate that an increased incorporation of

[35S]methionine

intoa small number of

DNA-binding

polypeptides

occurred as a result of a

temperature shift from the

nonpermissive

to

the

permissive

temperature. This increased in-corporation was found to occur in proteins

bind-ing tightly to

single-stranded

DNA, but not

double-stranded DNA, and was notdetected in

untransformed NRK cells subjected to

tempera-ture shift.

The procedure of

continuous

labeling in

[3H]leucine

and short-term labeling in

[35S]methionine

allows the 35S/3H ratios to be

used

as a means

of comparing

the amount of

recently synthesized

protein with the total

amount

synthesized for

individual

species of

protein. Alterations in this ratio may occur as a result of: (i) an altered rate of de novo synthesis

(transcriptional

or

translational

control), or (ii)

a decreased rate of degradation(proteolytic

con-trol). As yet, we have not determined which of

these

mechanisms is

responsible

for the

in-creased

incorporation of

[35S]methionine

into

single-stranded

DNA-binding proteins

during

transformation.

Most of the

DNA-binding

polypeptides

inthe

two

fractions

binding tightly

to

single-stranded

DNA

exhibited

increased 35S/3H ratios. If the

increased

incorporation

of

[35S]methionine

does

infactrepresent an increased

synthesis

ofthose

proteins, one explanation might be the

exis-tence of a coordinate control of the

synthesis

or

cellular

handling of

a group of proteins

with

similar

or

interrelated

functions. Another

ex-planation

might be that some of the

polypep-tides

that

appear in

the

gels

as

minor

bandsare

actually degradation products of

the

large

poly-peptide

of molecular

weight 93,000,

which

com-prises

more

than

60%

of the

protein

in that

function.

Most

of the

single-stranded

DNA-binding

proteins

of procaryotic

cells have been

impli-cated

in some aspect

of

DNA

replication

(8).

Therefore,

it is not

surprising that

an

increase

in

synthesis

of this

group

of proteins

precedes

or

coincides with the

onset

of

DNA

synthesis

in

shifted

ts339(RSV)NRK cells

as

demonstrated

in

this

paper.

We

are

currently investigating

the possibility that increased

synthesis

of these

proteins may occur as a

result

of the

onset

of

DNA

synthesis

and might

therefore be

inde-pendent

of transformation

perse.

Recently,

we

have

determined that the

93,000-dalton

poly-peptide

shows

an

increased

incorporation

of

[35S]methionine within h

1

after the

shift

to

[image:8.504.62.256.108.257.2]

the permissive

temperature

(in

preparation).

TABLE 3. Percentageof 35S remaining in labeling medium after1-hpulse ofts339(RSV)NRK cells

Labeling

temp0

35S

(final/initial)b

(_C)

39 0.62

33 0.63

aCultures were labeled for 1 h at either 39 or

330C.

Those cultures labeled at

330C

had been shifted from 39to330C 5 hbeforelabeling was be-gun.

h35S was determined by withdrawing aportion ofmediumcontaining [35S]methionine for scintilla-tion counting before labeling cells (initial 35S) and afterlabeling cells for 1 heither at 39 or

330C

(final

35S). Experimental protocol was identical to that described inTable 2.

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0.60

2.0

39

33 _{39 33}

,-_

MW

R

-

2 50

1.91 -200 1.26

NI

7

0

1.70 r-H14

0

1.33 --115

0.81 ---i05

0

93 -96

1.07 -91

1.02

77

0.94 e.

*L_68

1.31 _-63

0.77 -6

0

1.14

a

...N

--53

0.80 m

-47

1.06

-

42

1.33

1.03

27

1.19 -23

1.10

'".

MW

R

-200

1.22 -150

1078

1-93

1.38 -72

-66

1.33

1.14 -59

0.83 -50

-45

43

41 W-35

0.85 1?57

1.08

1.1

9

1.

10

1.13 -26

0.74 --22

1.10

FIG. 5. PAGEelectropherograms of ts339(RSV)NRK DNA-bindingproteinsobtainedintheexperiment describedinTable 2. Theprofiles ofthe DNA-bindingproteinselatedfromdenaturedDNAwith0.60 MNaCl and 2.0 M NaClareshown, along with the R values andmolecular weights (x1O-30 MW) for the major correspondingpeaks. The molecular weights were estimatedfrom companiongels containing proteins of knownmolecular weights (see MaterialsandMethods).

Thisindicates that synthesis ofthispolypeptide

precedes DNA synthesis by several hours and

that it isoneof the earliestevents to be

corre-lated withtheonsetoftransformation.

Although we have used theterm

"cytoplas-micDNA-binding protein" throughout this

in-vestigation, it should be pointedout thatmany

of theproteins separated in this investigation

may be compartmentalized to the nucleus in

vivo. Hypotonic swelling of the cells, followed

__

ao

*1.

I

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R value

FIG. 6. Histogram ofRvaluesfor159 correspond-ingDNA-binding polypeptidesobtainedfromthe

ex-perimentdescribed in Table2withts339(RSV)NRK cells. AllDNA-bindingprotein fractions were

ana-lyzed byPAGE, afterwhichgels were sliced

trans-verselyfor scintillationspectrometry. R valueswere

obtainedforcorresponding peaks from shifted and nonshiftedcultures. Arrow denotesmean.

by Dounce

homogenization,

could be

responsi-ble

for the release

ofsoluble proteins from

nu-clear to

cytoplasmic

compartments.

However,

Choe and

Rose

(7) found thatatleasthalf of the

cytoplasmic

DNA-binding

proteins of WI-38

cellsweretransported into nuclei within1

h,

in

the

absence

of DNA

synthesis. These

results

would

indicate

that the

cytoplasmic

DNA-bind-ing

proteins

may

migrate

to

the nucleus

and

perform

DNA-related functions there. How-ever, itremainsto bedetermined whether the

proteins

whose synthesis

is

altered

after the

onsetoftransformation inthe present

investi-gation

do in fact have

DNA-related roles.

ACKNOWLEDGMENTS

WethankFrances Byrd for excellent technical

assist-ance. This workwas supportedby Public Health Service

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